The study was designed as a pilot study for a harvesting procedure that simultaneously allows both cell cultures and histological assessment from gingival biopsies of very reduced dimensions, in an attempt to minimize any possible disturbance of the healing process.
Patient selection
Biopsies from three different patients were included in this study. The subjects were selected from the outpatients of the Department of Periodontology of the Victor Babes University of Medicine and Pharmacy of Timisoara, Romania. They met the following inclusion criteria: at least 2 but not more than 4 adjacent inferior teeth (up to the 2nd premolar) with less than 2mm of mucosa that required augmentation; no need of root coverage at the time of grafting. The exclusion criteria employed for each patient were: active periodontal disease, smokers, patients with history of alcohol abuse, systemic diseases, known alergies, pregnacies, and acute or chronic infections. The patients’ background information was as follows: Patient 1 (Sample 1) - 38 yo, female, biopsy site: teeth – 3.2–4.3; Patient 2 (Sample 2)–52 yo, female, biopsy site: teeth 3.3–3.5; Patient 3 (Sample 3)–56 yo, female, biopsy site: teeth 4.3–4.5.
Clinical procedures
Prior to surgery, demographics, medical and dental histories were collected. An oral examination and professional cleaning were performed, clinical measurements were done, x-rays and photographs were taken, oral hygiene procedures reviewed and reinforced. Alveolar bone level and surgical position limits were obtained at baseline. The first follow-up visit occurred one week post-surgery and the second visit one week later. At each visit, biopsies were taken and sent to the cell culture laboratory for further analysis. The surgical procedure consisted of placing a fully resorbable 3D collagen matrix designed for soft tissue regeneration (Geistlich Mucograft®, Geistlich Pharma AG, Wolhusen, Switzerland) at the surgical site using a modification of a well-known protocol [10, 39]. Briefly, after local anesthesia, a coronal incision was made at the muco-gingival junction extending at least to the line angle of the adjacent teeth, and vertical incisions were made at both the mesial and distal aspects of the grafted sites, so that rectangular wound beds were slightly larger than the collagen matrix. A partial-thickness flap was performed, was displaced apically and was sutured with 6-0 resorbable sutures. Muscle fibers were removed to expose the periosteal bed. The collagen matrix was cut to fit the recipient site, was placed dry and was sutured in place with single non-resorbable and resorbable6-0 sutures disposed circumferentially, so that the matrix soaked with blood would stabilize the clot over the wound bed. Lips and cheek adjacent to the grafted sites were put under tension, to ensure there was no traction on the operated areas. (Figures 1 a-d). Patients were instructed to use chlorhexidine 0.12 % mouth rinse for 30 s twice daily, to avoid aggressive rinsing or brushing of the grafted area and hard foods for two weeks after the surgery. Sutures were removed after ten days. After two weeks, brushing was resumed using soft brushes and delicate movements to avoid any trauma. Normal brushing was resumed after six weeks.
Biopsy harvesting procedure
Following a protocol described in the literature [10], biopsies of full-depth mucosa (down to the bone level) from pristine keratinized gingival areas and newly formed keratinized gingiva were harvested under local anesthesia using a 3-mm biopsy punch, prior to surgery, after 7 and after 14 days, for a different histological study (data to be published). A part of each sample was used for cell cultures in the present study, the rest was used for further detailed histological analysis.
All biopsies were performed from the central zone of the grafted area under the dental operating microscope with the aid of microsurgical instruments to avoid any disturbance of the healing process. To determine the exact region of harvesting and to avoid harvesting twice from the same site, preoperative and postoperative photographs were taken and surgical sketches were drawn. Specimens were fixed in buffered 4 % formaldehyde and sent to the histology laboratory. The fixed biopsies were oriented in a colored-coded biomimetic gel (BiopsyBoat™, Themis Pathology SRL, Bucharest, Romania), post-fixed with formal calcium, dehydrated in graded ethanols, and embedded in celloidin-parrafin. Semi-serial sectioning was performed at 5 μm and the resulting sections were stained with hematoxilin-eosin (HE).
Immuno-magnetic isolation of oral keratinocyte progenitor cells
Cell culture protocols and cell separations were performed using a protocol described in detail by Calenic et al [40, 41]. Briefly, biopsies were rinsed with phosphate buffer saline at pH 7and subjected to enzymatic dissociation in Collagenase (Sigma, St. Louis, MO) and Dispase (Sigma, St. Louis, MO) at 40C overnight. Following primary culture, the cells were separated using MACS (Magnetic Activated Cell Sorting, MACS Miltenyi Biotec, Bergisch Gladbach, Germany) and two surface markers: CD71 and integrin α6β4 (Mouse monoclonal [450–30A] antibody to Integrin alpha 6 beta 4 (Abcam) conjugated with Fluorescein isothiocyanate (FITC); goat anti-mouse IgG MicroBeads and CD71 MicroBeads). After both separation steps, three cell fractions were obtained: α6β4neg, α6β4pos CD71pos and α6β4 pos CD71neg fraction. In the present study, we analyzed α6β4pos CD71pos fraction, which, in our previous studies, demonstrated important progenitor cells attributes. The cells were further grown in Petri dishes pre-coated with human collagen IV (Sigma) (20μg/ml).
Viability of oral keratinocyte progenitor cells
For oral keratinocyte progenitor cells, cellular viability was assayed using Trypan blue exclusion. Briefly, Trypan blue stains dead cells in blue; thus the number of dead blue cells among the total number of cells was counted. For statistical purposes the assay was performed five times.
Colony forming efficiency of oral keratinocyte progenitor cells
In order to evaluate colony forming efficiency, 1x104 cells were seeded on type IV-collagen as described above; after 14 days, the cells were fixed and stained with crystal violet. Colonies with more than 20 cells were counted with Cell Analyst (AssaySoft Inc., Fountain Valley, CA, USA). For statistical purposes the assay was performed five times.
Immunofluorescence of oral keratinocyte progenitor cells
Immunofluorescence staining techniques followed traditional well-established protocols. Thus, following isolation, the cells were fixed using 4 % paraformaldehyde and further permeabilized with Triton X 100, followed by staining using a selection of primary antibodies, and finally by labeled secondary antibodies (as described in detail in section Antibody Library). For negative controls, the primary antibody was omitted during the immunofluorescence staining procedure. All samples were further observed under a confocal scanning laser fluorescent microscope.
Cell size of oral keratinocyte progenitor cells
Photographs of OKSCs populations were taken using a light microscope, and the images were analyzed using Cell Analyst (AssaySoft Inc., Fountain Valley, CA, USA). The assay was repeated five times, with 20 cells being counted for each experiment.
Antibody library
For magnetic isolation, the following antibodies were used: MicroBeads conjugated to anti-human CD71 (Miltenyi Biotec, Inc., Auburn, CA, USA); mouse monoclonal [450-30A] antibody to integrin a6b4 (Abcam, Germany) and anti-mouse IgG MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA). For immunofluorescence staining the following antibodies were used: mouse monoclonal anti-p63 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); mouse monoclonal anti-cytokeratin (CK) 10 (Acris GmbH, Hertford, Germany); mouse monoclonal anti-CK14 (Sigma-Aldrich, Germany). Primary antibodies were diluted at 1: 200. Alexa Fluor—conjugated donkey anti-mouse (Invitrogen, Eugene, OR) was used as a secondary antibody. Nuclei staining was done with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Eugene, OR, USA).
T Lymphocytes isolation
Samples of 10ml of peripheral venous blood were collected on anticoagulant [Heparin 15000 IU/5mL, Biochemie GmbH, Kundl, Austria]. Separation of mononuclear cells (PBMCs—Peripheral Blood Mononuclear Cells) from peripheral blood samples was performed by centrifugation on Ficoll-Paque™ Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient. After centrifugation, the supernatant was removed, and the cell pellet was used for in vitro analysis or frozen at -80 °C and kept for further investigation. Part of the PBMCs obtained in the previous step were cultured in order to increase the population of T-lymphocytes for 48 h in a specific culture medium (T-Cell expansion Stemline media, Sigma-Aldrich), supplemented with 10 ml L-Glutamine 200 mM/500 ml medium. After 48 h of in vitro cultivation, the cells were placed in contact with the 3D collagen matrix (1 x 105 T cells/mm3 collagen matrix) and transferred to 24-well plates. The comparative analysis was performed between the cells in contact with the 3D collagen matrix and the control-group cells.
T Lymphocytes viability
For the determination of induction and apoptotic execution propensity, the Annexin-V/PI method was used. In order to distinguish apoptotic cells from cells with permeabilized plasma membrane during the late non-apoptotic death, a viability marker (propidium iodide) was used, so that only apoptotic cells appeared positive for annexin V, and double-positive cells (annexin V+ iodide Propidium+) were eliminated.
Immunophenotyping analysis of T-cells by flow-cytometry
Lymphocyte immunophenotyping analysis was carried out both for lymphocytes cultured in the main culture medium, as well as for lymphocytes which have been in contact with the collagen matrix for 5 days. Surface markers were evaluated by flow cytometry after preparing the cells for this purpose. The cells, with a 105 cells/ml concentration, were washed in PBS, resuspended in PBS and incubated for 30 min in the dark with fluorochrome-conjugated monoclonal antibodies using the manufacturers recommended dilutions. After washing with a dedicated solution (Cell Wash Solution, BD Biosciences, San Jose, CA, USA), the cells were resuspended in 500 μl Cell Wash and analyzed with a FACSCalibur flowcytometer (BD Biosciences, San Jose, CA, USA). Data acquisition was performed using the program CellQuest Pro software (BD Biosciences, San Jose, CA, USA), and data analysis using the free flow cytometry data analysis Flowing Software 2.5.
Statistical analysis
For the oral keratinocytes in vitro behaviour, results from five independent experiments are shown as means ± SD. For each independent experiment, the same number of cells was used. Statistical analysis was performed using Student’s t-test. Statistical significance was accepted at p < 0.05. For T-lymphocytes data, Student’s t-test was employed for all data.